title: Proposal of a quantitative PCR-based protocol for an optimal Pseudomonas aeruginosa detection in patients with cystic fibrosis.
abstract: BACKGROUND
  The lung of patients with cystic fibrosis (CF) is particularly sensitive to Pseudomonas aeruginosa. This bacterium plays an important role in the poor outcome of CF patients. During the disease progress, first acquisition of P. aeruginosa is the key-step in the management of CF patients. Quantitative PCR (qPCR) offers an opportunity to detect earlier the first acquisition of P. aeruginosa by CF patients. Given the lack of a validated protocol, our goal was to find an optimal molecular protocol for detection of P. aeruginosa in CF patients.
  
  
  METHODS
  We compared two formerly described qPCR formats in early detection of P. aeruginosa in CF sputum samples: a qPCR targeting oprL gene, and a multiplex PCR targeting gyrB and ecfX genes.
  
  
  RESULTS
  Tested in vitro on a large panel of P. aeruginosa isolates and others gram-negative bacilli, oprL qPCR exhibited a better sensitivity (threshold of 10 CFU/mL versus 730 CFU/mL), whereas the gyrB/ecfX qPCR exhibited a better specificity (90% versus 73%). These results were validated ex vivo on 46 CF sputum samples positive for P. aeruginosa in culture. Ex vivo assays revealed that qPCR detected 100 times more bacterial cells than culture-based method did.
  
  
  CONCLUSIONS
  Based on these results, we proposed a reference molecular protocol combining the two qPCRs, which offers a sensitivity of 100% with a threshold of 10 CFU/mL and a specificity of 100%. This combined qPCR-based protocol can be adapted and used for other future prospective studies.
authors.affiliation: EA 3882-Laboratoire de Biodiversité et d'Ecologie Microbienne (LUBEM), SFR 148 ScInBioS, Faculté de Médecine, Université de Brest, Brest F-29200, France. hery@univ-brest.fr.
authors.forename: Florence
authors.initials: F
authors.lastname: Le Gall
authors.forename: Rozenn
authors.initials: R
authors.lastname: Le Berre
authors.forename: Sylvain
authors.initials: S
authors.lastname: Rosec
authors.forename: Jeanne
authors.initials: J
authors.lastname: Hardy
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Gouriou
authors.forename: Sylvie
authors.initials: S
authors.lastname: Boisramé-Gastrin
authors.forename: Sophie
authors.initials: S
authors.lastname: Vallet
authors.forename: Gilles
authors.initials: G
authors.lastname: Rault
authors.forename: Christopher
authors.initials: C
authors.lastname: Payan
authors.forename: Geneviève
authors.initials: G
authors.lastname: Héry-Arnaud
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D003550:Cystic Fibrosis / Q000175:diagnosis / Q000382:microbiology*; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D011552:Pseudomonas Infections / Q000175:diagnosis / Q000382:microbiology*; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers